Stevia rebaudiana contains sweet tasting compounds, namely stevioside and rebaudioside A that are mainly used as calorie-free sweeteners. Steviol is the aglycone derivative of these compounds. Steviol can be used to improve cognitive functions; see e.g. WO 2009/071277 (DSM IP ASSETS, B.V.)
Production of steviol from steviosides is difficult. Acid hydrolysis of stevioside its troublesome because under acidic conditions, the steviol that is produced rearranges into isosteviol. There have even been some literature reports that steviol cannot be obtained through this method (Kohda et al 1976 Phytochemistry 15: 981-983.)
An alternative method for cleavage of steviosides to produce steviol using sodium periodate was described by Ogawa et al 1980; Tetrahedron 36: 2641-2648. However, this process calls for a highly diluted system and a large excess (about >10 mol equivalents) of the expensive sodium periodate to achieve useful yields. Thus, this process is not economic for producing large amounts of steviol.
There have been a few enzyme-based methods described:
Bridel et al 1931 Bull. Soc. Chimie Biol. 13:781-96 noted that stevioside was not hydrolyzed by a variety of “fermenting products” such as emulsin, rhamnodiastase, Aspergillus niger or macerated and air-dried bottom fermentation yeast. The authors were successful using diastase from the snail (Helix pomatia). However, they obtained “barely 0.5 cm3 of pure digestive juice” per animal, and thus is not appropriate for large scale production. Mosettig et al, 1955 J. Org. Chem. 20: 884-899 also used enzymatic hydrolysis of the glucosides using snail enzyme preparations refer to as “enzyme juice” and a “dry preparation” to produce small amounts (less than 0.5 grams) of steviol. No further characterization of the enzymes were given.
Khoda et al., supra reported that other authors used a crude hesperidinase to generate steviol from stevioside, but when this was repeated, rebaudioside B and glucose were obtained rather than the desired steviol.
Ruddat et al 1965 Arch. Biochem. Biophys. 110: 496-499; Gianfagna et al. 1983 Phytochemistry 22(2): 427-430; and Pezzuto et al. 1985 Proc. Natl. Acad. Sci. USA 82: 2475-2482 each described a procedure using various commercial pectinases (“Pectinol 59L” from Rohm & Haas; “Pectinol 50L” from Corning Biosystems; “Pectinol AC” from Rohm & Haas, respectively). Davis et al, 1992 Phytopathology 82: 1244-50 reported that “Pectinol 59L” was no longer commercially available, and it is not clear if the other pectinols are still sold.
There is a need for an efficient process of producing steviol from steviosides which is suitable for commercial, large scale production.